The invention relates to new tricyclic 2,3-benzodiazepine derivatives substituted by halogen atom and to pharmaceutical compositions containing the same.
Among the 2,3-benzodiazepines with dimethoxy or methylenedioxy substitution at the benzene ring several became known for their biological activity and therapeutic use. The Hungarian patent specifications Nos. 155,572, 179,018, 191,702 and 195,788 disclose 7,8-dimethoxy derivatives. These compounds exhibit primarily anxiolytic and/or antidepressant as well as positive inotropic effect. Compounds having methylenedioxy substitution at the same position of the benzene ring are disclosed in the Hungarian patent specifications Nos. 191,698, 191,702, 206,719, in the U.S. Pat. No. 5,459,137 and the published international patent application No. WO 96/04283.
Unlike the former compounds the 2,3-benzodiazepine derivatives substituted with a methylenedioxy group have mainly anticonvulsive, muscle relaxant and neuroprotective effect. In the literature it is widely known that the noncompetitive inhibition of the AMPA receptor constitutes the basis of the action of these compounds [S. D. Donevan et al.: Neuron 10, 51-59 (1993), J. Pharmacol. Exp. Ther. 271, 25-29 (1994), I. Tarnawa et al.: Bioorg. Med. Chem. Lett., 3, 99-104 (1993)].
It is known that in the central nervous system of mammals L-glutamic acid is the most important excitatory neurotransmitter. At pathological conditions the extracellular glutamic acid concentration is pathologically increased causing acute or chronic damage in the neurons of the central nervous system.
The effect of excitatory amino acids (such as glutamic acid) is exerted by the activation of the inotropic (ion channel) and G-protein bound metabotropic receptors. The types of ionotropic glutamate receptors were designated according to the agonists which are suitable for their selective excitation. Accordingly three types of receptors are differentiated: NMDA, AMPA and kainate (formerly quisqualate) receptors which are subdivided into further subgroups [Ann. Rev. Neurosci. 17, 31, (1994)].
It was confirmed that in several acute and chronic diseases where the central nervous system is involved, e. g. epilepsy, diseases with adjuvant muscle spasms and various neurodegenerative diseases, AMPA-type glutamate receptors are playing a major role, and anticonvulsive, muscle relaxant and neuroprotective effect may be achieved by inhibiting the AMPA receptors [Cerebrovasc. Brain Metab. Rev. 6, 225 (1994); Neurology 44, Suppl. 8, S14 (1994); J. Pharmacol. Exp. Ther. 260, 742 (1992)].
The inhibition of AMPA receptor activation may be attained with both competitive and noncompetitive antagonists. The use of noncompetitive antagonists may be generally more advantageous than that of competitive antagonists as they give a higher level of protection at extremely high endogenous concentrations of excitatory amino acids [Epilepsy Res., 15, 179 (1993)].
Based on the above it was an observation of major importance that the types of 2,3-benzodiazepines, substituted with a methylenedioxy group, described in the introduction, possess anticonvulsive, muscle relaxant and neuroprotective properties due to their noncompetitive AMPA antagonist effect and consequently can be used in therapy as anticonvulsive, antiepileptic agents in acute and chronic neurodegenerative diseases as well as potentially in all diseases where the inhibition of excitatory amino acids is desirable at receptor levels.
Research involving the synthesis and pharmacological investigation of novel 2,3-benzodiazepines designed for therapeutic use revealed that the novel 2,3-benzodiazepine derivatives according to the invention, substituted with halogen on the benzene ring and having a heterocyclic ring fused to the 7-membered ring, possess significant AMPA antagonistic effect and thus can be used for the treatment of the diseases of the central nervous system mentioned above. Furthermore it was found that the novel compounds according to the invention have more advantageous properties than the known compounds.
Based on the above the invention relates to novel 2,3-benzo-diazepines of general formula (I), their potential stereoisomers and acid addition salts, 
wherein
R1 and R2 represent independently hydrogen, halogen, a C1-4 alkyl, C1-4 alkoxy, nitro, trifluoromethyl group or a group of general formula NR8R9, wherein
R8 and R9 represent independently hydrogen, a C1-4 alkyl group or a group of general formula xe2x80x94COR10, wherein
R10 represents hydrogen, an optionally substituted C1-4 alkyl group, C6-10 aryl group, C1-4 alkoxy group, C3-5 cyclo-alkyl group, C2-6 alkenyl group, C3-5 cycloalkoxy group or a group of general formula xe2x80x94NR11R12, wherein
R11 and R12 represent independently hydrogen, a C1-4 alkyl group, C3-5 cycloalkyl group or C6-10 aryl group,
X represents hydrogen or chlorine atom,
Y represents chlorine or bromine atom,
A represents a group of general formula (a), (b), (c) or (d), 
wherein
R3, R4, R5, R6 and R7 represent independently hydrogen, a C1-4 alkyl group, C3-5 cycloalkyl group, C2-4 alkenyl group, C2-4 alkinyl group or C6-10 aryl group which can optionally be substituted by one or more halogen, nitro, alkoxy or amino groups; furthermore heteroaryl group; groups of general formula xe2x80x94COOR13 or xe2x80x94COxe2x80x94NR14R15, wherein
R13 represents hydrogen or C1-4 alkyl group,
R14 and R15 represent independently hydrogen or a C1-4 alkyl group or form together with the nitrogen atom a 5 to 7-membered saturated heterocycle which can contain further oxygen, sulfur or nitrogen atoms.
In the groups of general formula (I) the alkyl and alkenyl groups can be both straight and branched groups. The cycloalkyl group can be a cyclopropyl, cyclobutyl or cyclopentyl group. The aryl group can be a phenyl or naphthyl group. The heteroaryl group can be an aromatic heterocyclic group, e.g. thienyl, furyl, pyridyl, etc.
When compounds of general formula (I) have a chiral centrum, the term xe2x80x9cisomerxe2x80x9d represents both enantiomers, furthermore, due to stereoisomers developing because of particular substitutions, E and Z isomers, diastereomers, tautomers as well as mixtures thereof, e. g. racemates.
The salts of the compounds of general formula (I) are physiologically acceptable salts formed with inorganic and organic acids. Suitable inorganic acids are e.g. hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid. Suitable organic acids are formic acid, acetic acid, maleic and fumaric acid, succinic acid, lactic acid, tartaric acid, citric acid or methanesulfonic acid.
A preferred group of the compounds of general formula (I) of the invention are those of general formula (Ia) wherein X and Y, or at least Y represent a chlorine atom, R1 represents an amino group in position 4 and R2 stands for hydrogen, furthermore one of R3 or R4 stands for a methyl group.
Compounds of general formula (I) of the invention are prepared by
a) reacting a compound of general formula (II) or (III), 
xe2x80x83wherein R1, R2, X and Y have the same meaning as above and Z represents a C1-3 alkylthio group, with
xcex1) an aminoacetal or aminoketal of general formula (IV), 
xe2x80x83wherein R3 and R4 have the same meaning as above, R16 and R17 represent independently C1-4 alkyl group or together a C2-4 alkylene group,
the intermediate formed in the reaction is submitted to acidic ring closure resulting in a compound of general formula (I), wherein R1, R2, X and Y have the same meaning as above and (A) represents a group of general formula (a), wherein R3 and R4 have the same meaning as above, or
xcex2) an acid hydrazide of general formula (V) 
xe2x80x83wherein R5 has the same meaning as above, or the compounds of general formula (II) or (III) are first reacted with hydrazine hydrate and the resulting intermediates are treated with an acid anhydride, yielding compounds of general formula (I) wherein R1, R2, X and Y have the same meaning as above and (A) represents a group of general formula (b), wherein R5 has the same meaning as above, or
(b) in a compound of general formula (III), wherein R1, R2, X and Y have the same meaning as above and Z represents a hydroxymethyl group, this latter group is converted into an aminomethyl group which is acylated and closed to a ring, resulting in a compound of general formula (I) wherein (A) represents a group of general formula (c), wherein R6 has the same meaning as above, or
(c) a compound of general formula (III), wherein R1, R2, X and Y have the same meaning as above and Z represents a methyl group, is reacted with a compound of general formula (VI), 
xe2x80x83wherein R7 has the same meaning as above and Hal represents a chloro or bromo substituent, wherby a compound of general formula (I) is obtained, wherein (A) represents a group of general formula (d), wherein R7 has the same meaning as above,
then, if desired, in the compound of general formula (I) obtained by any of the alternative processes the nitro group is reduced or the amino group is acylated, alkylated, or via diazotization substituted by a halogen or hydrogen atom, thus transforming it into an other compound of general formula (I) and/or the stereoisomers are separated and optionally a salt is formed.
The 4-thioxo-2,3-benzodiazepines of general formula (II), used for preparing some of the compounds of the invention, are synthesized by thionating the corresponding 4-oxo-2,3-benzodiazepine derivatives, using advantageously phosphorus pentasulfide or Lawesson reagent, and conducting the reaction in pyridine. The preparation of 4-oxo-2,3-benzodiazepines is known from the literature [F. Gatta. et al.: I] Farmaco Ed. Sc. 40, 942 (1985) and A. Chimirri et al.: J. Med. Chem. 40, 1258 (1997)] and essentially the process reported therein was followed.
Eventually the preparation of the compounds of the invention can be advantageously realized by starting from compounds of general formula (III), wherein Z represents an alkylthio group. In a suitable process 4-(methylthio)-5H-2,3-benzodiazepine derivatives are applied as starting materials which are suitably prepared by methylating compounds of general formula (II). The methylation of compounds of general formula (II) is preferably carried out with e. g. methyliodide in acetone in the presence of an acid binding agent.
Compounds of general formula (I) wherein (A) represents a group of general formula (a) are prepared by condensing the corresponding 4-thioxo compound of general formula (II) in an organic solvent, e. g. ethyleneglycol monomethyl ether, with an xcex1-aminoacetal or -ketal of general formula (IV). The acetal or ketal group can be open or can have a ring structure. In the course of condensation the liberated sulfur is bound by a suitable reagent, preferably e. g. mercuri oxide or silver salts. The resulting intermediate compound is isolated and processed usually as a raw product in the ring closing reaction which is preferably realized by heating in a mixture of ethanol and hydrochloric acid.
The xcex1-aminoacetals or -ketals used as reagents are known in the literature and are prepared accordingly [Jiro Adachi et al.: J. Org. Chem. 37, 221 (1972); Skinzo Kano et al.: Heterocycles 26, 2805 (1987); Org. Synth. 64, 19 (1986)].
Compounds of general formula (I), wherein (A) represents a group of general formula (b), are advantageously prepared by reacting a compound of general formula (III), wherein Z represents a methylthio group, in an organic solvent, e. g. ethyleneglycol monomethyl ether, with an acyl hydrazide, in the presence of catalytic amounts of an acid, e.g. p-toluenesulfonic acid. In this case condensation and ring closure proceed in a single reaction step resulting in the corresponding triazolo-2,3-benzodiazepine.
The same compounds can also be prepared by reacting first the 4-methylthio-2,3-benzodiazepine derivative of general formula (III) with hydrazine hydrate or by a condensation reaction between the corresponding 4-thioxo-benzodiazepine and hydrazine hydrate in the presence of e.g. mercury oxide and the resulting 4-hydrazino-2,3-benzodiazepine derivative is reacted with the chosen acid anhydride yielding the expected triazolo-2,3-benzodiazepine.
Compounds of general formula (I) wherein (A) represents a group of general formula (c) or (d) can be prepared by starting from the corresponding 4-methyl-5H-2,3-benzodiazepines. The latter ones can be synthesized by analogous processes disclosed in the Hungarian patent specifications Nos. 179,018, 191,702, 194,529 and 194,550.
The compounds of general formula (I), wherein (A) represents a group of general formula (c), are advantageously prepared by converting the 4-methyl group of the corresponding 4-methyl-5H-2,3-benzodiazepine derivative to in aldehyde group e. g. by oxidation with selenium dioxide, then reducing the aldehyde group with sodium borohydride to a hydroxymethyl group. In the obtained compound of general formula (III), wherein Z stands for a xe2x80x94CH2OH group, the hydroxy group is converted into an amino group by the Mitsunobu reaction (O. Mitsunobu: Synthesis 1, 1981). Namely, the compound of general formula (III), wherein Z represents a hydroxymethyl group, is reacted under the known reaction conditions with phthalimide, the resulting phthalimidomethyl group is converted to the aminomethyl group by hydrazinolysis, or in the Mitsunobu reaction the hydroxymethyl compound is first transformed into the azidomethyl compound, then by methods known from the literature the azido group is reduced or treated with triphenylphosphine to yield the amino group. The ring closure of the compounds obtained after acylating the 4-aminomethyl-2,3-benzodiazepine derivatives is carried out preferably by a reaction with e. g. phosphorus oxychloride.
The compounds of general formula (I), wherein (A) means a group of general formula (d), are prepared by reacting the above 4-methyl-5H-2,3-benzodiazepine derivatives with a 3-halo-2-oxo-carboxylic acid ester of general formula (VI), e. g. ethyl bromo-pyruvic acid or xcex1-haloketones. The reactions are performed on the basis of analogue ring closing reactions known from the literature [e. g. C. Casagrande et al.: J. Med. Chem. 11, 765 (1968); C. Galera et al.: J. Het. Chem. 23, 1889 (1986); Y. Blache et al.: J. Het. Chem. 32, 1317 (1995)].
In the compounds of general formula (I) the reduction of the nitro group is usually carried out in polar solvents, at room temperature or at higher temperatures, in the presence of Raney-nickel, platinum or palladium catalysts. In addition to hydrogen gas hydrazine hydrate, ammonium formate or cyclohexene may serve as hydrogen sources. Optionally the amino group can be transformed further by known methods, e. g. alkylation, acylation or in a Sandmeyer reaction.
The AMPA antagonistic effect of the compounds of general formula (I) is confirmed by the following studies.
Inhibition of AMPA Receptors
Two experimental models were applied to demonstrate the inhibitory effect of the compounds of general formula (I) on AMPA receptor activation. In the first model the xe2x80x9cspreading depressionxe2x80x9d inducing effect of glutamate agonists was studied, while in the second model the transmembrane ion current induced by the activation of glutamate receptors was directly measured.
Inhibition of AMPA and Kainate Induced xe2x80x9cSpreading depressionxe2x80x9d in the Isolated Chicken Retina Preparation
The kainate and AMPA antagonizing effect was studied in vitro in the retinal xe2x80x9cspreading depressionxe2x80x9d model [M. J. Sheardown: Brain Res. 607, 189 (1993)]. The AMPA/kainate antagonists extend the latency of the kainate (5 xcexcM) or AMPA (5 xcexcM) induced development of xe2x80x9cspreading depressionxe2x80x9d.
In the chicken retina model the kainate induced xe2x80x9cspreading depressionxe2x80x9d was inhibited by the compounds of the invention at IC50 values of 0.5 and 5 xcexcM. The IC50 value of the compound of Example 17 amounted to 2.5 xcexcM while those of Examples 18 and 35 amounted to 0.5 and 0.98 xcexcM, resp. The response to AMPA could usually be inhibited slightly less and the major part of IC50 values was in the range of 3-15 xcexcM. Thus the AMPA induced xe2x80x9cspreading depressionxe2x80x9d was inhibited by the compound of Example 17 at an IC50 value of 7.3 xcexcM and by compounds of Examples 21 and 27 at IC50 values of 4.3 and 3.1 xcexcM, resp. This demonstrates that the compounds of the invention, beyond strongly inhibiting AMPA receptors, also inhibit an other non-NMDA type glutamate receptor group, the specific kainate receptors.
Inhibition of AMPA and Kainate Induced Transmembrane Currents
The effect of the compound of Example 17 on whole cell currents induced by 100 xcexcM kainate or 5 xcexcM AMPA, resp., was studied on Purkinje cells of the cerebellum according to the method of Bleakman et al. [Neuropharmacology 12, 1689 (1996)]. The IC50 value against kainate was 4.97 xcexcM and that against AMPA 2.02 xcexcM. In this preparation kainate was also exerting its effect through the activation of AMPA receptors. According to the obtained IC50 values the ion current induced by AMPA receptor activation is twice as strongly inhibited by the compound of Example 17 than by the reference compound GYKI 52466 having also a 2,3-benzodiazepine structure [5-(4-aminophenyl)-9H-1,3-dioxolo[4,5-h][2,3]-benzodiazepine; Hungarian Patent specification No. 191,698, Example 8], which had IC50 values of 8.8 and 11.0, resp.
Anticonvulsive Effect
In the therapy of epilepsy a great variety of drugs is used but unfortunately they have severe side effects, furthermore the disease exists in particular forms which fail to respond to the available drugs. Thus novel antiepileptic drugs are required with a mechanism of action which is different from that of the available drugs. The introduction of drugs acting on the central nervous system by lowering the overactivation by glutamate are awaited with great expectation [TIPS 15, 456 (1994)].
In Table I the anticonvulsive effect of some compounds of the invention in the electroshock test is presented [J. Pharmacol. Exp. Ther. 106, 319 (1952)]. The anticonvulsive effect was also studied in convulsions induced by various chemical agents, e. g. pentetrazole [J. Pharmacol. Exp. Ther 108, 168 (1953)], strychnine [J. Pharmacol. Exp. Ther. 129, 75 (1960)], bemegride, nicotine, bicuculline, 4-aminopyridine and 3-mercaptopropionic acid. The pretreatment period was 60 minutes. The test compounds were administered orally in 3 doses to 10 male CD 1 mice in each dose group. The results are presented in Table 1.
The above data demonstrate the significant anticonvulsive effect of the compounds of general formula (I) in the ES test. The compound of Example 17 has a broad spectrum anticonvulsive effect even compared to phenytoin, a drug widely used in therapy.
Muscle Relaxant Effect
In clinical practice central muscle relaxants are used when the tonicity of skeletal muscles is increased due to muscle injury, trauma of the spinal cord or brain, or some chronic degenerative disease, and hyperreflexia or tremor develops. Muscle spasms are often painful and inhibit normal motility.
The muscle relaxant activity of the compounds of general formula (I) was measured in the xe2x80x9cinclined screenxe2x80x9d test of Randall [J. Pharmacol. Exp. Ther. 129, 163 (1960)] and in the rotarod test [J. Am. Pharm. Assoc. 46, 208 (1975)]. The compounds were administered in 3 i. p. doses to ten CD 1 mice in each dose group. The muscle relaxant activity of the compounds of the invention was demonstrated by the results obtained with the compounds of Examples 17 and 27 (Table 2).
The efficacy of the compounds of general formula (I) in the above muscle relaxant tests demonstrates that they can have therapeutic value in treating diseases where increased muscular tone is the problem to be solved.
Inhibition of Focal Ischemia
The focal ischemia inhibiting effect of the compound of Example 17 was studied on the xe2x80x9cmiddle cerebral artery occlusionxe2x80x9d (MCAO) test in anesthesized rats. The blood supply of the arteria cerebri media was transitorily inhibited with an intralaminarly introduced embolus, then perfusion was reinstated by removing the embolus and thus a human xe2x80x9cstrokexe2x80x9d like status was induced in an experimental animal model, in rats. After histological processing the developed infarcted area was measured by a computerized scanner program [R. T. Bartus: Stroke 11, 2265 (1994) and S. G. Sydserff: Brit. J. Pharmacol. 114, 1631 (1995)]. The results obtained are presented in Table 3.
In the above experiment the rate of cerebral cell damage, induced by the occlusion of the middle cerebral artery, was significantly reduced by 6xc3x971 mg/kg i. v. doses of the compound of Example 17 in the best human stroke animal model.
Based on the above pharmacological results the compounds of general formula (I) according to the invention can influence the dysfunctions of the AMPA receptors. Thus the compounds according to the invention are suitable for the treatment of neurological and psychiatric disorders induced by the extremely increased activation of the AMPA receptor. Consequently in therapy they can be applied as muscle relaxants, anticonvulsive and neuroprotective agents. They possess therapeutic value in the treatment of epilepsy, diseases associated with spasms of the skeletal muscles, acute and chronic neurodegenerative disorders, e. g. cerebral ischemia (stroke).
Neurological diseases which can be prevented or treated in this way are the following: Parkinson""s disease, Alzheimer""s disease, Huntington chorea, amyotrophic lateral sclerosis, olivopontocerebellar atrophy, AIDS dementia and senile dementia. They are also suitable for the treatment of neurodegenerative states developed as a result of cerebrovascular disasters (stroke, cerebral and spinal traumas), hypoxia, anoxia or hypoglycemic states, resp. The compounds of the invention can be advantageously applied for the treatment of various psychiatric diseases, e. g. anxiety, schizophrenia, sleeping disorders, alleviation of symptoms of alcohol, drug and narcotics withdrawal. They can be beneficial in preventing the development of tolerance against sedative drugs and pain killers.
They are assumed to be suitable drugs in epileptic diseases, in the treatment of muscle spasms of central origin and in the alleviation of pathological pain.
For therapeutic use the compounds of general formula (I) of the invention can be converted into enteral or parenteral pharmaceutical compositions. For this purpose organic and inorganic carriers and auxiliary materials are employed, e. g. water, gelatin, acacia gum, lactose, starch, magnesium stearate, talc, vegetable oil, polyethyleneglycols, etc.
The pharmaceutical composition can be prepared in solid form, e.g. tablet, coated tablet, suppository or capsule form or also in liquid form, e. g. solution, suspension or emulsion form. The above auxiliary materials can be also supplemented with other additives, such as preservatives, stabilizing, emulsifying agents, buffers, etc.
For parenteral use the active ingredient is prepared in the form of sterile solution or suspension. The sterile vehicle can contain in addition adjuvants such as a local anesthetic agent, stabilizing agent or buffer, resp. The dosage of the active ingredient depends on the route of administration, the type and severity of the disease as well as the mass and age of the patient. The daily dose can be 0.5-1000 mg, preferably 20-200 mg, in a single dose or divided in several doses.